Modification of chromatin structure via the reversible acetylation of nucleosomal histones plays an important role in the regulation of transcription in eukaryotic cells. Acetylation of the C-amino group of specific lysine residues within the amino terminal tail of core histones results in localized chromatin relaxation. This acetylation is required to maintain the unfolded structure of nucleosomes undergoing transcription. In general, histone acetylation activity correlates with transcriptional activation, whereas deacetylation activity correlates with transcriptional repression. The regulation of histone acetylation levels in vivo is a dynamic process under the control of competing enzymes within the nucleus of a cell: histone acetyltransferases (HATs) and histone deacetylases (HDACs).
Currently, over a dozen HATs have been identified and at least 18 different members of the HDAC family have been isolated from mammalian cells. The first cDNA encoding HDAC to be cloned was the yeast protein Rpd3, which was identified in genetic screens for transcriptional repressors. Mammalian HDAC1 was cloned independently as the molecular target of trichostatin A (TSA), a fungal toxin. HDAC1 was observed to be an ortholog of yeast Rpd3, and both were shown to have HDAC activity in vitro.
HDACs are usually separated into 3 classes on the basis of their similarity to various yeast histone deacetylases: (i) class I members, including HDAC1, HDAC2, HDAC3, HDAC8, and HDAC11, which are homologous to the yeast Rpd3 protein 5-11 (ii) class II HDACs, including HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, which have similarities to yeast Hda1 and, (iii) nicotinamide adenine dinucleotide (NAD)-dependent sirtuin (SIRT) proteins, which are homologous to the yeast Sir2 protein. Up to now, 7 human SIRT homologues have been identified.
Class I HDACs have approximately 350-500 amino acids and their transcript expression is considered ubiquitous. Class II HDACs are much larger proteins with around 1000 amino acids; their mRNA distribution is more restricted and they are implicated in the development and differentiation of cardiac and skeletal striated muscle. Class II enzymes can shuttle in and out of the nucleus upon certain cellular signals. Among class I members, HDAC1 and HDAC2 are localized exclusively in the cell nucleus while HDAC3 can be detected in the nuclear and cytoplasm compartments. Database searches for expressed sequence tags showing high similarity with class I HDACs has lead to the cloning of the cDNA for human HDAC8, the fourth identified class I HDAC. This enzyme encodes 377 amino acid residues and is evolutionary most similar to HDAC3 with 34% overall identity.
HDACs mediate transcriptional repression by interacting with larger multisubunit complexes. For example, HDAC1 is known to bind the corepressor Sin3, and HDAC1-Sin3 further associates with the silencing mediators NCoR and SMRT. N-CoR/SMRT-HDAC1 is then recruited by specific transcription factors bound to promoter elements within the nucleus. For example, the retinoblastoma (Rb) gene product recruits N-CoR/SMRT-HDAC1 to bind the transcription factor E2F to repress E2F-regulated promoters. HDAC1-Sin3 also binds to and mediates repression by the MAD/MAX repressor heterodimer. The histone deacetylation activity of HDAC1 is essential for this transcriptional repression.
Recently, it has become clear that a number of non-histone nuclear proteins, such as the tumor suppressor p53, are also substrates for histone deacetylases, which regulate their activity by deacetylation. In addition, some HDACs, such as HDAC6 and SIRT2, are prominently expressed in the cell cytoplasm; these histone deacetylases have been shown to deacetylate a non-histone cytosolic protein: the cytoskeletal protein α-tubulin with concomitant destabilization of dynamic microtubules. Finally, recent reports have suggested a previously unrecognized HDAC location in the cell. Indeed, it has been demonstrated that SIRT3, a human SIR2 homologue, is a mitochondrial NAD-dependent deacetylase, suggesting that this sirtuin may deacetylate a substrate localized in this organelle.
There is a need in the art for methods of treating smooth muscle cell disorders. The present invention addresses this need.